Project Summary from Original Application The remarkable spatial and temporal precision of Ca2+ signals finely controls a vast array of cellular functions. Ca2+ signals reflect the function of highly coordinated Ca2+ sensors and Ca2+ channels. The interaction between Ca2+ sensor STIM proteins in the ER and Orai Ca2+ channels in the PM, is crucial in Ca2+ signal generation. The two proteins physically couple in ER-PM junctions, mediating ?store-operated? Ca2+ signals, essential in controlling gene expression, secretion, motility and growth. Their role in Ca2+ signaling is critical, and defects in STIM or Orai proteins cause a spectrum of disorders including severe combined immunodeficiency, muscular hypotonia, autoimmunity, skin dysplasia, and exocrine defects. Dysregulation of STIM/Orai expression is closely linked to cardiovascular and airway remodeling, neurodegenerative disorders, altered immunity, and cancer. We have defined much on the molecular properties and cellular organization of STIM and Orai proteins. We now turn toward the nanoscale ER-PM junctional environment in which they operate. Junctional Ca2+ is critical in generating both ?local? Ca2+ signals and in sustaining ?global? Ca2+ oscillations that extend across the cytoplasm and penetrate the nucleus. Using a compendium of molecular probes, imaging technology, gene-deleted cellular systems, and animal knockout models, our work is directed toward three major areas: (1) Identifying critical differences in the disease-related STIM and Orai isoforms, focusing on operation of the widely expressed STIM2.1 splice variant of STIM2 that lacks the critical Orai-binding domain; using super-resolution STED microcopy and TIRF/FRET imaging we explore a model by which STIM2.1 disrupts the cross-linking of Orai channel subtypes within junctions, and determine how clustering organizes the junctional Ca2+ signaling machinery. (2) Defining the micro-physiological environment of Ca2+ entry junctions, utilizing genetically encoded and optogenetically applied Ca2+ probes tagged onto STIM and Orai proteins to monitor the highly restricted junctional Ca2+ micro-environment; using such measurements to explore how STIM-mediated clustering of Orai channels controls local junctional Ca2+ signals, modifies the proximity of InsP3Rs and SERCA pumps, and modulates the generation of global Ca2+ oscillations. (3) Understanding how store-operated Ca2+ signals are transcriptionally transduced, exploring how the ?STIM2 phenotype? in smooth muscle- and B cell-targeted STIM1-deleted animals is related to STIM1/STIM2.1 expression levels and junctional Ca2+ signals, how STIM- modulated Orai clustering controls specific NFAT subtype activation, and determining transcriptome changes related to STIM type-specific Ca2+ signal generation. Our studies address the critical gap in our knowledge of how junctional Ca2+ tunes the generation of local and global Ca2+ signals. The central role of STIM/Orai-mediated store-operated Ca2+ signals in cell physiology, the widespread dysfunction caused by STIM/Orai mutations, the deep functional distinctions between STIM/Orai subtypes, and the extraordinary alteration of subtype expression in major disease states, all define the STIM-Orai interface as a pivotal therapeutic target.